For some time, there has been a move to develop an artificial skin that can be used (1) for wound healing and the repair of ulcerated, burned or lacerated skin, and (2) as a model to test substances for irritation, toxicity, inflammation and pharmacology so as to reduce the number of tests using live animals. This latter application for artificial skin is commonly referred to as an in vitro alternative to animal testing.
Berg and colleagues (U.S. Pat. Nos. 4,703,108 and 4,970,298, the disclosures of which are herein incorporated by reference) have developed a biocompatible, chemically crosslinked, three-dimensional collagen matrix as a substitute for the dermal layer of the skin where it promotes fibroblast ingrowth and proliferation when implanted into animals (Doillon, et al., "Fibroblast-collagen sponge interactions and the spatial deposition of newly synthesized collagen fibers in vitro and in vivo," J. Scanning Electron Microscopy, III:1313-1320 (1984). It has also been used to treat human Decubitus ulcers where it promotes healing (Silver, F. H., et al., "Collagenous materials enhance healing of chronic skin ulcers," Biomedical Materials and Devices Research Society, 110:371-376 (1989)). In vitro studies, this matrix has been used as a model for examining the role of various matrix components on fibroblast ingrowth (Doillion, et al., "Fibroblast growth on a porous collagen sponge containing hyaluronic acid and fibronectin," Biomaterials 8:195-200 (1987).
In this invention, this matrix is used as a support for human keratinocyte growth and differentiation. The matrix described herein, containing keratinocytes and fibroblasts, is referred to herein as a "skin model system" or "SMS." Dollion, et al., in "Behavior of fibroblasts and epidermal cells cultivated on analogues of extracellular matrix," Biomaterials, 9:91-96 (1988), report on efforts to use the Berg collagen matrix in attempts to manufacture artificial skin, but such attempts were not successful. Epidermal cells on the surface of the matrix were neither differentiated nor in stratified layers.
An alternative collagen-based system has been developed by Bell et al. (Bell, E., et al., "The reconstitution of living skin," Journal of Investigative Dermatology, 81:2s-10s (1983); Bell, E., et al., "Recipes for reconstituting skin," J. Biomechan. Eng., 113:113-119 (1991)) as a dermal replacement called "living skin equivalent" or "LSE". The LSE is manufactured by mixing living human fibroblasts with soluble rat tail collagen under conditions where the collagen forms a gel (See, U.S. Pat. Nos. 4,485,096, 4,604,346, 4,8356,102, and Bell, E., et al., "Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro," Proc. Natl. Acad. Sci. USA, 76:1274-1278 (1979)). During the five days of culture, the gel containing fibroblasts undergoes a contraction process where the collagen volume is reduced to a small disc approximately 10% to 20% of the original volume depending on the concentration of collagen, the cell number and the composition of the growth medium. This contracted collagen matrix is then used to support human keratinocyte growth. Although the Bell LSE is an advance over other previously known artificial skin systems, it does suffer from disadvantages. Since the manufacture or the collagen matrix requires living fibroblasts, it is expensive to manufacture and the matrix is not easily stored. The Bell collagen matrix is not cross-linked and contracts with the addition of the fibroblasts, so it is difficult to manufacture the matrix in a desired shape and size. It is difficult to make large sizes of LSE; the matrix contracts substantially and large numbers of living cells are required. The Bell collagen matrix utilizes soluble collagen, which is more difficult and expensive to extract than insoluble collagen. Still further, since the Bell collagen matrix requires living cells, the manipulations to which it can be exposed are limited, e.g., it cannot be exposed to toxic conditions which might manipulate or favorably alter the matrix structure but which would kill the cells. In view of cheese limitations, there remains a need for improved artificial skin systems.